PXD003929

PXD003929 is an original dataset announced via ProteomeXchange.

Title	Identification and Characterization of MCM3 as a KEAP1 Substrate
Description	KEAP1 is a substrate adaptor protein for a CUL3-based E3 ubiquitin ligase. Ubiquitylation and degradation of the antioxidant transcription factor NRF2 is considered the primary function of KEAP1; however, few other KEAP1 substrates have been identified. Because KEAP1 is altered in a number of human pathologies and has been proposed as a potential therapeutic target therein, we sought to better understand KEAP1 through systematic identification of its substrates. Towards this goal, we combined parallel affinity capture proteomics and candidate-based approaches. Substrate-trapping proteomics yielded NRF2 and the related transcription factor NRF1 as KEAP1 substrates. Our targeted investigation of KEAP1 interacting proteins revealed MCM3, an essential subunit of the replicative DNA helicase, as a new substrate. We show that MCM3 is ubiquitylated by the KEAP1-CUL3-RBX1 complex in cells and in vitro. Using ubiquitin remnant profiling, we identify the sites of KEAP1-dependent ubiquitylation in MCM3, and these sites are on predicted exposed surfaces of the MCM2-7 complex. Unexpectedly, we determined that KEAP1 does not regulate total MCM3 protein stability or subcellular localization. Our analysis of a KEAP1 targeting motif in MCM3 suggests MCM3 is a point of direct contact between KEAP1 and the MCM hexamer. Moreover, KEAP1 associates with chromatin in a cell cycle-dependent fashion with kinetics similar to the MCM2-7 complex. KEAP1 is thus poised to affect MCM2-7 dynamics or function rather than MCM3 abundance. Together, these data establish new functions for KEAP1 within the nucleus and identify MCM3 as a novel substrate of the KEAP1-CUL3-RBX1 E3 ligase.
HostingRepository	PRIDE
AnnounceDate	2017-10-12
AnnouncementXML	Submission_2017-10-12_00:06:56.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Dennis Goldfarb
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	ubiquitination signature dipeptidyl lysine; phosphorylated residue; acetylated residue; iodoacetamide derivatized residue
Instrument	LTQ Orbitrap Velos; LTQ Orbitrap Elite

Revision	Datetime	Status	ChangeLog Entry
0	2016-04-08 05:46:29	ID requested
⏵ 1	2017-10-12 00:06:57	announced

Mulvaney KM, Matson JP, Siesser PF, Tamir TY, Goldfarb D, Jacobs TM, Cloer EW, Harrison JS, Vaziri C, Cook JG, Major MB, Identification and Characterization of MCM3 as a Kelch-like ECH-associated Protein 1 (KEAP1) Substrate. J Biol Chem, 291(45):23719-23733(2016) [pubmed]

curator keyword: Biological

submitter keyword: HEK293T, SILAC, label free quantification, Di-Gly, APMS

Michael Benjamin Major
contact affiliation	Lineberger Comprehensive Cancer Center, Dept of Cell Biology & Physiology, UNC Chapel Hill
contact email	ben_major@med.unc.edu
lab head
Dennis Goldfarb
contact affiliation	Cell and Developmental Biology
contact email	dennisg@email.unc.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD003929
     1. Label: PRIDE project
     2. Name: Identification and Characterization of MCM3 as a KEAP1 Substrate